UCSF

Though the outputs of regulatory circuits are conserved over long timescales, the exact mechanisms of regulation change comparatively frequently. One such example is the regulation of cell type in yeast, specifically the haploid specific genes. These are transcribed in both of the mating competent cell types, a and alpha, and not in the diploid a/alpha cell type. The simplest and likely ancestral mode of regulation is direct repression of the haploid specific genes by the Mata1-Matalpha2 heterodimer in the a/alpha cell. However, this is not the only solution. Here we discuss two examples where the output of the circuit has been maintained but the molecular mechanism is different in the regulation of haploid specific genes in yeast. After bioinformatic searches indicated the lack of a Mata1-Matalpha2 site in GPA1—one of the haploid specific genes in Lachancea kluyveri—further inspection revealed a tripartite Mata1-Matalpha2-Mcm1 site in GPA1. ChIPseq of Matalpha2 and a reporter experiments testing the tripartite site confirmed that this gene is directly repressed by tripartite Mata1-Matalpha2-Mcm1, while confirming that the other haploid specific genes are repressed by Mata1-Matalpha2. Models made from existing structural data further supported that the three proteins could bind the tripartite site to co-repress GPA1. This depends on an ancestral gain of a domain on Matalpha2 that enables interaction with Mcm1. In the other example, in the species Wickerhamomyces anomalous, a lack of evidence for Mata1-Matalpha2 binding in the upstream regions of all haploid specific genes—except the transcription factor Rme1— indicated that Mata1-Matalpha2 regulation might be indirect for these genes. We knocked out Rme1, and by assaying the effect on mating and transcriptionally profiling the haploid specific genes with RNAseq, we found that two of the haploid specific genes are activated by Rme1. Further bioinformatic analysis suggests that this is direct regulation by Rme1. This is similar to indirect haploid specific gene regulation via Rme1 in another species, K. lactis, indicating that this likely happened more than once, and that Rme1’s ancestral regulation by Mata1-Matalpha2 positioned it to acquire this new role in regulating haploid specific genes. In both examples, transcriptional regulators already associated with the transcriptional circuit gained a new regulatory role with a few cis changes in target genes. Together, these examples illustrate how changes in regulatory circuits can build on each other to create new regulatory architectures, adding to our overall understanding of how transcriptional regulation shifts over time.